The mechanism of radiation repair and biochemical basis of cellular sensitivity in mammalian cells are poorly understood. Our long term goal is to identify genes and gene products in these processes and to study their mode of action at the molecular level. This proposal illustrates the initial goal of isolating and characterizing the cDNAs involved in radiation sensitivity. Initially, a wild type of Chinese hamster ovary (CHO) cell line and its X-ray sensitive mutants (xrs) will be used as the model system. At a later stage, the relevance between those cDNAs and the radiation sensitivity in human tumors will be studied. Seventy-two bacterial clones containing wild type specific cDNAs were isolated by the technique of differential hybridization. The mRNAs of those cDNA molecules are absent or poorly expressed in the xrs-5 mutant. The lack of one or some of those mRNAs is responsible for the xrs phenotype. The ability to synthesize those mRNAs in the revertant appears to be the mechanism of reversion of the xrs mutation. Colony hybridization, using the mRNAs of revertants as the probe to screen the 72 clones, can rapidly eliminate those cDNAs not related to radiosensitivity. cDNAs showing a positive signal will be further tested by hybridization against mRNAs from another mutant, xrs-6. CDNAs/mRNAs missing in both mutants will be the prime candidates for the functional assay. The cDNAs will be expressed in xrs mutants using an SV40-based vector. Both transient and permanent expression using G418 resistance as the selection marker will be used. A Cell Analyzer system will be used to monitor the radiation response of the cells transiently expressing the cDNA after isolated by flow cytometry. The effect of radiation on cell survival, double strand break rejoining and colony forming ability will be measured. Those cDNAs showing a corrective effect of the xrs mutation will be fully characterized by Northern hybridization and nucleotide sequence analysis. Finally, the level of expression of those cDNAs/mRNAs in the human tumor cell lines with different radiosensitivity will be studied. Results of this proposal will provide information on the molecular mechanism of radiation repair and sensitivity. The cDNAs may be able to serve as the marker for prediction of radiation responsiveness of human tumors.